Cell-matrix mechanical interactions drive fundamental processes such as developmental morphogenesis, wound healing, and the organization of bioengineered tissues. The overall goal of this research is to determine the underlying cellular and molecular mechanisms that regulate these critical biophysical processes in corneal fibroblasts, which should ultimately lead to more effective approaches to directing their biomechanical behavior in vivo and in vitro. In the first funding period, we developed a new experimental model for directly investigating cell-matrix mechanical interactions inside 3-D fibrillar collagen matrices. Data obtained using this innovative approach has provided new insights into potential mechanisms regulating sub-cellular force generation, matrix reorganization, as well as the modulation of cell behavior by mechanical stress which together lead to the following Hypotheses: 1) The balance between Rac and RhoA activity plays a central role in regulating the mechanical behavior of corneal fibroblasts inside 3-D matrices at the sub- cellular level. These effects are mediated by dynamic changes in cytoskeletal and focal adhesion organization, as well as differential regulation of myosin light chain (MLC) phosphorylation, 2) Corneal fibroblasts actively respond to increases or decreases in local matrix stress (including that produced by neighboring cells); these responses are mediated by compensatory and reciprocal changes in Rho and Rac activation, and 3) The pattern and amount of collagen matrix reorganization can be modulated by altering the balance between Rho and Rac activity, and is enhanced by cell-cell mechanical interactions. To test these hypotheses, we propose the following Specific Aims: 1) Determine the role of Rho and Rac in regulating cytoskeletal organization, mechanical behavior, and sub-cellular force generation by corneal fibroblasts inside 3-D matrices using our time-lapse imaging system, 2) Measure the dynamic mechanical response of corneal fibroblasts to changes in ECM stress; and, 3) Directly assess the process of cell-induced 3-D collagen matrix reorganization (alignment of collagen fibrils), and the roles of Rho and Rac in regulating this process. These studies will be the first to assess the roles of Rho and Rac on the subcellular pattern of force generation and ECM reorganization within 3-D collagen matrices. Overall, this research should provide unique insights into the mechanisms controlling corneal fibroblast migration, contraction, and matrix reorganization," critical ] processes in the.fields qfdevelopmental biology, wound healing and tissue engineering.